Hydrophobic interaction chromatography has been employed to show that the Ca2+ binding protein, calmodulin (CaM), associates with certain of its target enzymes through a Ca2+ induced hydrophobic binding region. Studies established that hydrophobic binding either is reinforced by electrostatic attractions or opposed by electrostatic repulsions to create a degree of specificity in the binding of calmodulin to certain proteins with accessible hydrophobic regions. These studies have advance the use of hydrophobic interaction chromatography to purify proteins and to study protein-protein and protein-membrane hydrophobic interaction. Ca2+ dependent hydrophobic interaction chromatography with phenyl-Sepharose has been used to rapidly and quantitatively purify CaM from various rat tissues, and from cells in culture, as an initial step prior to quantitation. The small amounts of CaM isolated in this manner from both cytosolic and Triton X-100 solubilized particulate fractions than can be determined directly with a protein assay. This quantitation procedure is more reliable and accurate than previously established assays for CaM, and allows the estimation of cytosolic and membrane-bound CaM.